Polymer comprising a plurality of phenothiazine groups and methods of making the same

ABSTRACT

A non-leaching mediator may include a polymer having a polymeric backbone, and a plurality of phenothiazine groups bonded to the polymeric backbone. The plurality of phenothiazine groups may include at least one of a phenothiazine group having the general formula (IV): 
                         
and salts thereof, where n is about 9 and “R” represents the polymeric backbone to which the phenothiazine group is bonded, and a phenothiazine group having the general formula (V):
 
                         
and salts thereof, where n is about 9 and “R” represents the polymeric backbone to which the phenothiazine group is bonded.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/334,814 filed Oct. 26, 2016 now U.S. Pat. No. 9,944,725, which is adivisional of U.S. patent application Ser. No. 14/963,908 filed Dec. 9,2015 and issued as U.S. Pat. No. 9,505,884, U.S. patent application Ser.No. 14/963,908 is a divisional of U.S. patent application Ser. No.14/259,408 filed Apr. 23, 2014 and issued as U.S. Pat. No. 9,238,717,U.S. patent application Ser. No. 14/259,408 is a divisional of U.S.patent application Ser. No. 13/116,427 filed May 26, 2011 and issued asU.S. Pat. No. 8,742,063, U.S. patent application Ser. No. 13/116,427claims the benefit of U.S. Provisional Application No. 61/349,469entitled “Polymer Bonded Redox Molecules And Methods Of Making The Same”filed May 28, 2010, all of which are incorporated by reference in theirentireties.

BACKGROUND

Biosensors usually analyze a sample of a biological fluid, such as wholeblood, urine, or saliva. Samples are compositions that may contain anunknown amount of analyte. Typically, a sample is in liquid form and isan aqueous mixture. A sample may be a derivative of a biological sample,such as an extract, a dilution, a filtrate, or a reconstitutedprecipitate. A biosensor usually determines the concentration of one ormore analytes, a substance present in the sample, such as ketones,glucose, uric acid, lactate, cholesterol, or bilirubin. An analysisdetermines the presence and/or concentration of the analyte in thesample. The analysis is useful in the diagnosis and treatment ofphysiological abnormalities. For example, a diabetic individual may usea biosensor to determine the glucose level in blood for adjustments todiet and/or medication.

A biological fluid may be obtained using a variety of methods. In oneexample of an invasive method, a lancet is used to pierce a user's skinto draw a biological fluid sample, such as blood. This sample is thenanalyzed with a biosensor external to the skin to determine theconcentration of an analyte, such as glucose, in the sample. Onedisadvantage of this method is that the user's skin must be pierced eachtime an analyte concentration reading is desired.

One alternative to such an invasive method is to implant a biosensorunder the user's skin. This method can allow for multiple analyteconcentration readings to be obtained without making a new puncture inthe skin for each reading. In addition, the analyte concentration may bemonitored at regular intervals without any action required by the user.Thus, implantable biosensors may offer improvements in user complianceand in the amount of information provided.

Many biosensors measure an electrical signal to determine the analyteconcentration in a sample of the biological fluid. The analyte typicallyundergoes an oxidation/reduction (redox) reaction when an excitationsignal is applied to the sample. A redox reaction includes oxidation andreduction half-cells. The oxidation half-cell of the reaction involvesthe loss of at least one electron by the first species. The reductionhalf-cell involves the addition of at least one electron to the secondspecies. The ionic charge of a species that is oxidized is made morepositive by an amount equal to the number of electrons removed.Likewise, the ionic charge of a species that is reduced is made lesspositive by an amount equal to the number of electrons gained.

In electrochemical sensor systems, a test excitation signal initiatesthe redox reaction of the analyte in the sample of the biological fluid.The test excitation signal usually is an electrical signal, such as acurrent or potential, and may be constant, variable, or a combinationthereof such as when an AC signal is applied with a DC signal offset.The test excitation signal may be applied as a single pulse or inmultiple pulses, sequences, or cycles. The redox reaction generates atest output signal in response to the excitation signal. The outputsignal usually is another electrical signal, such as a current orpotential, which may be measured and correlated with the concentrationof the analyte in the sample. The output signal may be measuredconstantly or periodically during transient and/or steady-state output.Various electrochemical processes may be used such as amperometry,coulometry, voltammetry, gated amperometry, gated voltammetry, and thelike.

An enzyme or similar species may be used to enhance the redox reactionof the analyte. The enzyme may be an analyte specific enzyme, such asglucose oxidase or glucose dehydrogenase, which catalyze the oxidationof glucose in a whole blood sample.

A mediator may be used to maintain the oxidation state of the enzyme. Amediator is a substance that may be oxidized or reduced and that maytransfer one or more electrons. A mediator is a reagent and is not theanalyte of interest, but provides for the indirect measurement of theanalyte. More simply, the mediator undergoes a redox reaction inresponse to the oxidation or reduction of the analyte. The oxidized orreduced mediator then undergoes the opposite reaction at an electrodeand is regenerated to its original oxidation number.

The mediator in an electrochemical biosensor may be a one electrontransfer mediator or a multi-electron transfer mediator. One electrontransfer mediators are chemical moieties capable of taking on oneadditional electron during the conditions of the electrochemicalreaction. One electron transfer mediators include compounds, such as1,1′-dimethyl ferrocene, ferrocyanide and ferricyanide, andruthenium(III) and ruthenium(II) hexaamine. Multi-electron transfermediators are chemical moieties capable of taking on more than oneelectron during the conditions of the reaction. Multi-electron transfermediators include two electron transfer mediators, such as the organicquinones and hydroquinones, including phenanthroline quinone;phenothiazine and phenoxazine derivatives;3-(phenylamino)-3H-phenoxazines; phenothiazines; and7-hydroxy-9,9-dimethyl-9H-acridin-2-one and its derivatives. Twoelectron transfer mediators also include the electro-active organicmolecules described in U.S. Pat. Nos. 5,393,615; 5,498,542; and5,520,786.

Two electron mediators may have redox potentials that are at least 100mV lower, more preferably at least 150 mV lower, than ferricyanide. Twoelectron transfer mediators include 3-phenylimino-3H-phenothiazines(PIPT) and 3-phenylimino-3H-phenoxazines (PIPO). Two electron mediatorsalso include the carboxylic acid or salt, such as ammonium salts, ofphenothiazine derivatives. Two electron mediators further include(E)-2-(3H-phenothiazine-3-ylideneamino)benzene-1,4-disulfonic acid(Structure A), (E)-5-(3H-phenothiazine-3-ylideneamino)isophthalic acid(Structure B), ammonium(E)-3-(3H-phenothiazine-3-ylideneamino)-5-carboxybenzoate (Structure C),and combinations thereof. The structural formulas of these mediators arepresented below. While only the di-acid form of the Structure A mediatoris shown, mono- and di-alkali metal salts of the acid are included. Thesodium salt of the acid may be used for the Structure A mediator.Alkali-metal salts of the Structure B mediator also may be used.

One drawback to the use of implantable electrochemical biosensors isthat one or more of the reagents of the biosensor may be released intothe biological sample during the analysis. Thus, one or more of thereagents, such as a mediator, may leach from the biosensor into thebodily fluid of the user. Leaching of reagents from the biosensor overtime can result in decreased accuracy of the readings obtained from thebiosensor. In addition, the reagents may cause undesirable physiologicaleffects if they are released into the patient at a level or rate that istoo large.

Mediators bonded to polymers have been investigated as possiblenon-leaching mediators in electrochemical biosensors; however, thesesystems have met with mixed success. Polymer bonded mediators may haveinsufficient reactivity in a redox reaction in response to the oxidationor reduction of the analyte. Some polymer bonded mediators includetransition metals, which could be harmful if released into the bodilyfluid of the user.

Accordingly, it would be desirable to have reagents for electrochemicalbiosensors that do not substantially leach into the biological fluid ofthe patient. Preferably such non-leaching mediators would be effectivein transferring electrons between the analyte and the electrodes and/orin maintaining the oxidation state of the enzyme.

SUMMARY

In one aspect, the invention provides a polymer that includes apolymeric backbone and a plurality of phenothiazine groups bonded to thepolymeric backbone. The plurality of phenothiazine groups includes atleast one of a phenothiazine group having the general formula (IV):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, and a phenothiazinegroup having the general formula (V):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded.

In another aspect, the invention provides a method of making the abovepolymer. The method includes forming the polymer from a polymer having aprecursor polymeric backbone and a plurality of nucleophilic side groupsbonded to the precursor polymeric backbone, and a mixture of2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid and2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

In another aspect, the invention provides a method of making the abovepolymer. The method includes forming the polymer from a compound havinga plurality of epoxide groups, and a mixture of2-(8-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid and2-(2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

In another aspect, the invention provides a polymer having the generalformula (VI):

and salts thereof; where n is about 9, x and y independently are aninteger from 1 to 100,000, and z is an integer from 0 to 100,000.

In another aspect, the invention provides a method of making the abovepolymer. The method includes forming the polymer frompoly(β-carboxyl-γ-(2-mercaptoethyl)carbamoyl-butene), and a mixture of2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid and2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

In another aspect, the invention provides a polymer having the generalformula (VII):

and salts thereof; where n is about 9, x and y independently are aninteger from 1 to 10,000, and z is an integer from 0 to 10,000.

In another aspect, the invention provides a method of making the abovepolymer. The method includes forming the polymer frompoly(diallylmethylamine), and a mixture of2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid and2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

In another aspect, the invention provides a polymer having the generalformula (VIII):

and salts thereof, where R′ is an organic group, where m is 0 or 1,where n is about 9, and where x and y independently are an integer from1 to 10,000.

In another aspect, the invention provides a method of making the abovepolymer. The method includes forming the polymer from a diepoxyalkane,and a mixture of2-(8-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid and2-(2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

The scope of the present invention is defined solely by the appendedclaims and is not affected by the statements within this summary.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale and are not intended to accurately representmolecules or their interactions, emphasis instead being placed uponillustrating the principles of the invention. Moreover, in the figures,like referenced numerals designate corresponding parts throughout thedifferent views.

FIGS. 1A and 1B depict graphs illustrating output currents from mediatorreduction in response to an input potential.

FIG. 2 depicts a graph illustrating output currents from mediatorreduction in response to an input potential when the mediator is in amixture containing glucose and glucose dehydrogenase.

FIG. 3 depicts a method for making a composition including at least onecompound having the general formula (I) or (II).

FIG. 4 depicts chemical structures, reaction schemes and product yieldsfor a method of making a composition including a mixture of a firstcompound having the general formula (I), and a second compound havingthe general formula (II), where X is bromine and each compound ispresent as the disodium salt.

FIGS. 5A and 5B depict graphs illustrating output currents from mediatorreduction in response to an input potential.

FIG. 6 depicts a method for making a compound having the general formula(III).

FIG. 7 depicts chemical structures and reaction schemes for a method ofmaking a compound having the general formula (III), where X is bromineand the compound is present as the disodium salt.

FIGS. 8A and 8B depict graphs illustrating output currents from mediatorreduction in response to an input potential.

FIG. 9 depicts a method for making a polymer.

FIGS. 10A and 10B depict chemical structures and reaction schemes formethods of making a composition that includes a polymer having thegeneral formula (VI) (FIG. 10A) or a polymer having the general formula(VII) (FIG. 10B).

FIG. 11 depicts a method for making a method for making a compositionincluding at least one compound having the general formula (IX) or (X),and a method for making a polymer.

FIG. 12 depicts chemical structures and reaction schemes for a method ofmaking a composition that includes a polymer having the general formula(VIII).

FIG. 13 depicts examples of polymer bondable mediators having terminalfunctional groups other than halogen.

FIG. 14 depicts examples of polymerizable mediators having terminalfunctional groups other than halogen.

DETAILED DESCRIPTION

A non-leaching mediator includes a compound or a mixture of compoundsthat is not substantially released into a biological sample, but thatmay be oxidized or reduced, and may transfer one or more electrons fromthe sample to an electrode of a biosensor. A non-leaching mediator alsoincludes a polymer having a polymeric backbone and a plurality offunctional groups bonded to the polymeric backbone, where the functionalgroups may be oxidized or reduced, and may transfer one or moreelectrons from the sample to an electrode of a biosensor.

The non-leaching mediator has sufficient solubility in the sample toprovide for the indirect measurement of the analyte, undergoing a redoxreaction in response to the oxidation or reduction of the analyte. Theoxidized or reduced mediator responsive to the analyte concentration ofthe sample then undergoes the opposite redox reaction at the workingelectrode of the biosensor and is regenerated to its original oxidationnumber. A measuring device may correlate the electrons flowing throughthe working electrode with the analyte concentration of the sample.

A non-leaching mediator may have sufficient solubility to provide forthe indirect measurement of the analyte, even if the entire mediatordoes not dissolve fully in the sample. For example, a non-leachingmediator may include a functional group that may be oxidized or reduced,and this functional group may be solubilized by the sample while atleast a portion of the remainder of the mediator is not solubilized inthe sample.

A non-leaching mediator compound may have the general formula (I):

and salts thereof, where n is about 9, X is a halogen, and X ispreferably bromine.

The term “salts thereof” means a compound in which the —H atoms of oneor both of the —SO₃H groups is replaced with a cation independentlyselected from the group consisting of alkali metal ions, alkaline earthmetal ions and ammonium ions. The term “halogen” means —F, —Cl, —Br or—I.

A non-leaching mediator compound may have the general formula (II):

and salts thereof, where n is about 9, X is a halogen, and X ispreferably bromine.

A non-leaching mediator composition may include a mixture of a firstcompound having the general formula (I) and salts thereof, and a secondcompound having the general formula (II) and salts thereof, where X ispreferably bromine.

FIGS. 1A and 1B depict graphs illustrating output currents from mediatorreduction in response to an input potential. The mediator was acomposition including a 3:2 molar mixture of a first compound having thegeneral formula (I), and a second compound having the general formula(II), where X is bromine and each compound is present as the disodiumsalt. A 5 mm glassy carbon electrode served as a working electrode (WE),Ag/AgCl as a reference electrode (RE), and platinum gauze as a counterelectrode (CE). In FIG. 1A, the mediator composition was present at aconcentration of 1 mg/mL in a mixture containing 10 mM PBS buffer (pH7.4), and the input potential was scanned between −200 mV and 200 mV vs.Ag/AgCl. In FIG. 1B, the mediator composition was present at aconcentration of 1 mg/mL in a mixture containing 100 mM PBS (pH 7.0) and100 mM NaCl buffer solution, and the input potential was scanned between−100 mV and 200 mV vs. Ag/AgCl. The rate of change of the inputpotential was varied from 10 mV/s to 800 mV/s, as indicated in FIGS. 1Aand 1B. Referring to FIG. 1B, when the scan rate was less than 50 mV/s,the oxidative and reductive peak separation was around 30-40 mV. Thisseparation indicates that the reduction of this composition was atwo-electron process, which is close to the theoretical limit ofNernstian behavior at 60 mV/2e.

The redox potential of the mediator composition of FIGS. 1A and 1B wasabout −3 mV vs. Ag/AgCl in the 10 mM PBS buffer (pH 7.4), and was about40 mV vs. Ag/AgCl in the mixture of 100 mM PBS (pH 7.0) and 100 mM NaClbuffer. These redox potentials are similar to that of the conventionalmediator (E)-2-(3H-phenothiazine-3-ylideneamino)benzene-1,4-disulfonicacid (Structure A), which is −50 mV vs. Ag/AgCl.

The open circuit voltage between the WE and the RE also was measured forthe mediator composition of FIGS. 1A and 1B. The open circuit voltage isa measure of the redox state of the mediator. The open circuit voltageof the mixture was 59 mV vs. Ag/AgCl in the 10 mM PBS buffer, whichsuggests that the mediator was in its oxidized state after synthesis.

FIG. 2 depicts a graph illustrating output currents from mediatoroxidation in response to an input potential when the mediator is in amixture containing glucose and glucose dehydrogenase. The mediator wasthe composition used in FIGS. 1A and 1B. A 5 mm glassy carbon electrodeserved as a WE, Ag/AgCl as a RE, and platinum gauze as a CE. Thecomposition was present at a concentration of 2 mg/mL in a mixturecontaining 10 mM PBS buffer (pH 7.4), and the input potential was 100 mVvs. Ag/AgCl. The output current increased as the glucose concentrationincreased from 0 to 1.2 mM in the mixture. Thus, the composition may beused as a mediator for the redox reaction correlating the glucoseconcentration in a sample with an electrical signal.

FIG. 3 depicts a method 300 of making a composition including at leastone compound having the general formula (I) or (II) and salts thereof.The method 300 includes forming 301 a ω-haloalkyl acetate 324 from anα-hydroxyl-ω-haloalkane 320 and acetic anhydride 322; forming 302 a(ω-acetoxyalkyl) triphenylphosphonium bromide 328 from the ω-haloalkylacetate 324 and triphenyl phosphine 326; forming 303 aω-(4-chloro-3-nitrophenyl)alk-ψ-enyl acetate 334 from the(ω-acetoxyalkyl) triphenylphosphonium bromide 328,4-chloro-3-nitrobenzaldehyde 330 and a base 332; forming 304 aω-(4-(2-bromophenylthio)-3-nitrophenyl)alk-ψ-enyl acetate 340 from theω-(4-chloro-3-nitrophenyl) alk-ψ-enyl acetate 334, 2-bromobenzenethiol336 and a base 338; forming 305 aω-(3-amino-4-(2-bromophenylthio)phenyl)alk-ψ-enyl acetate 346 from theω-(4-(2-bromophenylthio)-3-nitrophenyl)alk-ψ-enyl acetate 340, iron 342and ammonium chloride 344; forming 306 aω-(ωH-phenothiazin-2-yl)alk-ψ-enyl acetate 354 from theω-(3-amino-4-(2-bromophenylthio)phenyl)alk-ψ-enyl acetate 346, copperiodide 348, copper 350 and a base 352; forming 307 aω-(ωH-phenothiazin-2-yl)alkyl acetate 360 from theω-(ωH-phenothiazin-2-yl)alk-ψ-enyl acetate 354, molecular hydrogen 356and palladium-carbon 358; forming 308 aω-(ωH-phenothiazin-2-yl)alkan-α-ol 366 from theω-(ωH-phenothiazin-2-yl)alkyl acetate 360, an alcohol 362 and an acid364; forming 309 a 2-(ω-haloalkyl)-ωH-phenothiazine 372 from theω-(ωH-phenothiazin-2-yl)alkan-α-ol 366, carbon tetrahalide 368 andtriphenyl phosphine 370; and forming 310 a mixture of2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 376 and2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 378 from the 2-(ω-haloalkyl)-ωH-phenothiazine 372,2-aminobenzene-1,4-disulfonic acid 373 and Na₂S₂O₈ 374. Products 376 and378 are examples of compounds having the general formula (I) and (II),respectively.

FIG. 4 depicts chemical structures, reaction schemes and product yieldsfor a method 400 of making a composition including a mixture of a firstcompound having the general formula (I), and a second compound havingthe general formula (II), where X is bromine and each compound ispresent as the disodium salt. Method 400 includes forming 9-bromononylacetate 424 from 9-halononan-1-ol 420 and acetic anhydride 422; forming(9-acetoxynonyl)triphenylphosphonium 428 from 9-bromononyl acetate 424and triphenyl phosphine 426; forming10-(4-chloro-3-nitrophenyl)dec-9-enyl acetate 434 from(9-acetoxynonyl)triphenylphosphonium 428, 4-chloro-3-nitrobenzaldehyde430 and sodium bis(trimethylsilyl)amide (NaHMDS) 432; forming10-(4-(2-bromophenylthio)-3-nitrophenyl)dec-9-enyl acetate 440 from10-(4-chloro-3-nitrophenyl)dec-9-enyl acetate 434, 2-bromobenzenethiol436 and K₂CO₃ 438; forming10-(3-amino-4-(2-bromophenylthio)phenyl)dec-9-enyl acetate 446 from10-(4-(2-bromophenylthio)-3-nitrophenyl)dec-9-enyl acetate 440, iron 442and ammonium chloride 444; forming 10-(10H-phenothiazin-2-yl)dec-9-enylacetate 454 from 10-(3-amino-4-(2-bromophenylthio)phenyl)dec-9-enylacetate 446, copper iodide 448, copper 450 and K₂CO₃ 438; forming10-(10H-phenothiazin-2-yl)decyl acetate 460 from10-(10H-phenothiazin-2-yl)dec-9-enyl acetate 454, molecular hydrogen 456and palladium-carbon 458; forming 10-(10H-phenothiazin-2-yl)decan-1-ol466 from 10-(10H-phenothiazin-2-yl)decyl acetate 460, ethanol 462 andHCl 464; forming 2-(10-bromodecyl)-10H-phenothiazine 472 from10-(10H-phenothiazin-2-yl)decan-1-ol 466, carbon tetrabromide 468 andtriphenyl phosphine 470; and forming a mixture of2-(8-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 476 and2-(2-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 478 from the 2-(10-bromodecyl)-10H-phenothiazine 472,2-aminobenzene-1,4-disulfonic acid 473 and Na₂S₂O₈ 474. Products 476 and478 are examples of compounds having the general formula (I) and (II),respectively.

A non-leaching mediator compound may have the general formula (III):

and salts thereof, where n is about 8, X is a halogen, and X ispreferably bromine.

FIG. 5A and FIG. 5B depict graphs illustrating output currents frommediator reduction in response to an input potential. The mediator was acompound having general formula (III), where X is bromine. A 5 mm glassycarbon electrode served as a WE, Ag/AgCl as a RE, and platinum gauze asa CE. In FIG. 5A, the mediator was present at a concentration of 1 mg/mLin a mixture containing 10 mM PBS buffer (pH 7.4), and the inputpotential was scanned between −200 mV and 200 mV vs. Ag/AgCl. In FIG.5B, the mediator was present at a concentration of 1 mg/mL in a mixturecontaining 100 mM PBS (pH 7.0) and 100 mM NaCl buffer solution, and theinput potential was scanned between −100 mV and 200 mV vs. Ag/AgCl. Therate of change of the input potential was varied from 10 mV/s to 800mV/s, as indicated in FIGS. 5A and 5B. Referring to FIG. 5B, when thescan rate was less than 50 mV/s, the oxidative and reductive peakseparation was around 30-40 mV. This separation indicates that thereduction of this mediator was a two-electron process, which is close tothe theoretical limit of Nernstian behavior at 60 mV/2e.

The redox potential of the compound having general formula (III), whereX is bromine, was about 3 mV vs. Ag/AgCl in the 10 mM PBS buffer (pH7.4), and was about 15 mV vs. Ag/AgCl in the mixture of 100 mM PBS (pH7.0) and 100 mM NaCl buffer. These redox potentials are similar to thatof the conventional mediator(E)-2-(3H-phenothiazine-3-ylideneamino)benzene-1,4-disulfonic acid(Structure A), which is −50 mV vs. Ag/AgCl.

The open circuit voltage between the WE and the RE also was measured forthe compound having general formula (III), where X is bromine. The opencircuit voltage of the compound was 111 mV vs. Ag/AgCl in the 10 mM PBSbuffer, which suggests that the mediator was in its oxidized state aftersynthesis.

Referring to FIG. 2, a compound having general formula (III), where X isbromine, can be used as a mediator for the redox reaction correlatingthe glucose concentration of a sample with an electrical signal. A 5 mmglassy carbon electrode served as a WE, Ag/AgCl as a RE, and platinumgauze as a CE. The mediator was present at a concentration of 3 mg/mL ina mixture containing 10 mM PBS buffer (pH 7.4), and the input potentialwas 100 mV vs. Ag/AgCl. The output current increased as the glucoseconcentration increased from 0 to 1.2 mM in the mixture. Thus, thecomposition may be used as a mediator for the redox reaction correlatingthe glucose concentration in a sample with an electrical signal.

FIG. 6 depicts a method 600 of making a compound having the generalformula (III) and salts thereof. The method 600 includes forming 60110,10a-dihydro-3H-phenothiazin-8-ol 624 from8-methoxy-10,10a-dihydro-3H-phenothiazine 620 and an acid 622; forming602 a 8-(ω-haloalkoxy)-10,10a-dihydro-3H-phenothiazine 630 from10,10a-dihydro-3H-phenothiazin-8-ol 624, a α,ω-dihaloalkane 626 and abase 628; and forming 603 a2-(2-(ω-haloalkoxy)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 638 from 8-(ω-haloalkoxy)-10,10a-dihydro-3H-phenothiazine 630,2-aminobenzene-1,4-disulfonic acid 632, Na₂S₂O₈ 634 and a base 636.Product 638 is an example of a compound having the general formula(III).

FIG. 7 depicts chemical structures and reaction schemes for an exampleof a method 700 of making a compound having the general formula (III),where X is bromine and the compound is present as the disodium salt.Method 700 includes forming 10,10a-dihydro-3H-phenothiazin-8-ol 724 from8-methoxy-10,10a-dihydro-3H-phenothiazine 720 and HCl 722; forming8-(8-bromooctanoxy)-10,10a-dihydro-3H-phenothiazine 730 from10,10a-dihydro-3H-phenothiazin-8-ol 724, 1,8-dibromooctane 726 andpotassium carbonate 728; and forming2-(2-(8-bromooctanoxy)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 738 from 8-(8-bromooctanoxy)-10,10a-dihydro-3H-phenothiazine 730,2-aminobenzene-1,4-disulfonic acid 732, Na₂S₂O₈ 734 and sodium hydroxide736. Product 738 is an example of a compound having the general formula(III).

A non-leaching mediator polymer may include a polymeric backbone and aplurality of functional groups bonded to the polymeric backbone, wherethe functional groups may be oxidized or reduced. In one example, theplurality of functional groups may include a plurality of phenothiazinegroups bonded to the polymeric backbone. A phenothiazine group bonded toa polymeric backbone may include at least one of a phenothiazine grouphaving the general formula (IV):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, and a phenothiazinegroup having the general formula (V):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded.

Preferably the plurality of functional groups are bonded to thepolymeric backbone through carbon-heteroatom bonds. If the plurality offunctional groups includes a phenothiazine group having formula (IV) or(V), the phenothiazine group preferably is bonded to the polymericbackbone through an ether group, an ester group, a carbonate group, aurea group, a urethane group, an amine group, an amide group, athioether group, a thiourea group or a sulfonamide group.

A non-leaching mediator polymer that includes a polymeric backbone and aplurality of functional groups bonded to the polymeric backbone, wherethe functional groups may be oxidized or reduced, may include a polymerhaving the general formula (VI):

and salts thereof, where n is about 9, where x and y independently arean integer from 1 to 100,000, and where z is an integer from 0 to100,000.

Preferably, the sum of x, y, and z in the polymer having the generalformula (VI) is from 100 to 100,000. More preferably the sum of x, y,and z is from 500 to 10,000. Preferably x and y independently are aninteger from 10 to 2,000. More preferably x and y independently are aninteger from 20 to 1,000, and more preferably are an integer from 40 to500. Preferably the mole fraction of x and y is from 0.05 to 0.9, wherethe mole fraction of x and y is calculated as (x+y)/(x+y+z). Morepreferably the mole fraction of x and y is from 0.07 to 0.7, and morepreferably is from 0.1 to 0.5.

A non-leaching mediator polymer that includes a polymeric backbone and aplurality of functional groups bonded to the polymeric backbone, wherethe functional groups may be oxidized or reduced, may include a polymerhaving the general formula (VII):

and salts thereof, where n is about 9, where x and y independently arean integer from 1 to 10,000, and where z is an integer from 0 to 10,000.

Preferably, the sum of x, y, and z in the polymer having the generalformula (VI) is from 10 to 10,000. More preferably the sum of x, y, andz is from 20 to 1,000. Preferably x and y independently are an integerfrom 1 to 70. More preferably x and y independently are an integer from2 to 20, and more preferably are an integer from 30 to 10. Preferablythe mole fraction of x and y is from 0.05 to 0.9. More preferably themole fraction of x and y is from 0.07 to 0.7, and more preferably isfrom 0.1 to 0.5.

FIGS. 8A and 8B depict graphs illustrating output currents from mediatorreduction in response to an input potential. The mediator was present asa polymer having structure (VI) (FIG. 8A) or (VII) (FIG. 8B). For eachpolymer, the mole fraction of x and y was presumed to be 0.1. A 5 mmglassy carbon electrode served as a working electrode (WE), Ag/AgCl as areference electrode (RE), and platinum gauze as a counter electrode(CE). In FIG. 8A, the mediator composition was present at aconcentration of 0.6 mg/mL in a mixture containing 10 mM PBS buffer (pH7.4). In FIG. 8B, the mediator composition was present at aconcentration of 0.504 mg/mL in a mixture containing 10 mM PBS buffer(pH 7.4). The concentrations of the polymers in their respectivecompositions were selected to provide approximately the same moles ofphenothiazine groups in each composition. The mixtures were purged withnitrogen for 20 minutes before analysis using cyclic voltammetry. Theinput potential was scanned between −250 mV and 250 mV vs. Ag/AgCl. Therate of change of the input potential was varied from 10 mV/s to 100mV/s, as indicated in FIGS. 8A and 8B.

Referring to FIG. 8A, the redox peak potential for the mediatorcomposition including a polymer having structure (VI) was about +31 mVvs. Ag/AgCl. The oxidative and reductive peak separation was about 31.5mV. This separation indicates that the reduction of this composition wasa two-electron process, which is close to the theoretical limit ofNernstian behavior at 60 mV/2e. The diffusion coefficient for thepolymer was calculated to be about 3.5×10⁻⁸ cm²/s, for both the oxidizedand reduced forms of the functional groups.

Referring to FIG. 8B, the redox peak potential for the mediatorcomposition including a polymer having structure (VII) was about +71 mVvs. Ag/AgCl. The oxidative and reductive peak separation was about 66mV. This larger separation relative to that of the theoretical limit ofNernstian behavior may indicate some irreversibility in the oxidationand reduction of the functional groups of structure (VII). The diffusioncoefficient for the polymer having reduced functional groups wascalculated to be about 7×10⁻⁸ cm²/s, and the diffusion coefficient forthe polymer having oxidized functional groups was calculated to be about4×10⁻⁸ cm²/s.

A non-leaching mediator polymer that includes a polymeric backbone and aplurality of functional groups bonded to the polymeric backbone, wherethe functional groups may be oxidized or reduced, may include a polymerhaving the general formula (VIII):

and salts thereof, where R′ is an organic group, where m is 0 or 1,where n is about 9, and where x and y independently are an integer from1 to 10,000.

Preferably, the sum of x and y in the polymer having the general formula(VIII) is from 10 to 10,000. More preferably the sum of x and y is from20 to 1,000. Preferably x and y independently are an integer from 1 to70. More preferably x and y independently are an integer from 2 to 20,and more preferably are an integer from 30 to 10.

Preferably m is 1, and R′ is an organic group in the polymer having thegeneral formula (VIII). The R′ group may include an aliphatic group,such as a methyl group, an ethyl group, a propyl group, a butyl group,1,4-butanedioxy group, a 1,3-butanedioxy group, a1,4-cycloheanedimethoxy group, a 1,3-dioxy-2-propanol group, adiethylene glycoxy group, a neopentyl glycoxy group, anα,ω-oxy-poly(ethylene glycol) group, or an α,ω-oxy-poly(propyleneglycol) group. The R′ group may include an aryl group, such as abisphenol A group, a resorcinol group, a bisphenol A propoxy group, or abis-(4-oxyphenyl) group. The R′ group may include a siloxane group, suchas an α,ω-2-ethoxy-poly(dimethylsiloxane) group. The R′ group mayinclude a mixture of these groups, and more than one type of R′ groupmay be present in the polymer. Preferably the R′ group is an alkyl grouphaving from 1 to 10 carbon atoms, such as a methyl group, an ethylgroup, a propyl group, or a butyl group. More preferably, the R′ groupis an alkyl group having from 1 to 6 carbon atoms or from 1 to 4 carbonatoms, and more preferably is an ethyl group.

A non-leaching mediator polymer may be made by reacting one or morecompounds having general formula (I), (II) and/or (III) with a polymerhaving a precursor polymeric backbone and a plurality of nucleophilicgroups bonded to the precursor polymeric backbone. Examples ofnucleophilic groups that may be bonded to the precursor polymericbackbone include thiol groups, cyano groups, alcohol groups, carboxylategroups, amino groups and amido groups.

Examples of polymers having a precursor polymeric backbone and aplurality of nucleophilic groups bonded to the precursor polymericbackbone include polymers and copolymers formed from monomers containingnucleophilic groups, including N-thioethyl acrylamide, N,N′-cystaminebisacrylamide, cyanoacrylate, acrylonitrile, vinyl alcohol,hydroxystyrene, vinyl acetate, acrylic acid, methacrylic acid,aminoethyl methacrylate, vinyl pyridine, 4-vinylpyridine-N-oxide,allylamine, ethyleneimine, diallylmethylamine, lysine and/or acrylamide.Copolymers formed from the above monomers preferably are formed frommixtures of the monomers containing nucleophilic groups and monomerswithout the nucleophilic groups. Examples of polymers having a precursorpolymeric backbone and a plurality of nucleophilic groups bonded to theprecursor polymeric backbone also include cellulose and its derivatives,including carboxymethyl cellulose, ethyl cellulose and celluloseacetate. Presently preferred polymers having a precursor polymericbackbone and a plurality of nucleophilic groups bonded to the precursorpolymeric backbone includepoly(β-carboxyl-γ-(2-mercaptoethyl)carbamoyl-butene),poly(diallyl-methylamine) hydrochloride, 4-vinylpyridine-N-oxide,poly(allylamine) and poly(ethyleneimine).

FIG. 9 depicts a method 900 of making a polymer. The method 900 includesforming 902 a polymer having a polymeric backbone and a plurality ofphenothiazine groups bonded to the polymeric backbone 930, from apolymer having a precursor polymeric backbone and a plurality ofnucleophilic groups bonded to the precursor polymeric backbone 910, anda mixture of2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 920 and2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 922. The method 900 optionally includes forming 901 a polymerhaving a precursor polymeric backbone and a plurality of nucleophilicgroups bonded to the precursor polymeric backbone 910. The polymer 930may be, for example, a polymer having the general formula (VI) or apolymer having the general formula (VII).

Reactants 920 and 922 correspond to compounds 376 and 378 of FIG. 3,respectively. Preferably the2-(8-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 920 is a2-(8-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid, and more preferably is2-(8-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 476 of FIG. 4). Preferably the2-(2-(ω-haloalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 922 is a2-(2-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid, and more preferably is2-(2-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 478 of FIG. 4).

FIG. 10A depicts chemical structures and reaction schemes for a method1000 of making a composition including a polymer having the generalformula (VI). Method 1000 includes formingpoly(β-carboxyl-γ-(2-mercaptoethyl)carbamoyl-butene) 1010 frompoly(ethylene-alt-maleic anhydride) 1012 and 2-aminoethanethiolhydrochloride 1014. The polymer 1010 includes thiol groups (—SH) as theplurality of nucleophilic groups bonded to the precursor polymericbackbone. Method 1000 further includes forming polymer 1030 from amixture of the poly(β-carboxyl-γ-(2-mercaptoethyl)carbamoyl-butene)1010,2-(8-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1020 and2-(2-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1022. Reactants 1020 and 1022 are examples of compounds 376 and 378of FIG. 3, respectively.

In one example, 2-aminoethanethiol hydrochloride 1014 (5.50 g of 90%pure material, 43.8 mmol) was suspended in ether (50 mL) under anitrogen atmosphere. A solution of triethylamine (39.7 mmol, 4.01 g,5.53 mL) in ether (20 ml) was added dropwise to the suspension overfifteen minutes to form a first reaction mixture. The first reactionmixture was stirred overnight and was then filtered. The filteredmixture was added to a solution of poly(ethylene-alt-maleic anhydride)1012 (PEMA; molecular weight of 100,000-500,000 daltons, 5 g, 40 mmol)in acetone (70 mL) to form a second reaction mixture. The mixture wasstirred overnight before removing the precipitate by filtration. Theprecipitate was washed with acetone (50 ml), stirred in fresh acetone(50 ml) for ten minutes and filtered. The solid was dried in a vacuumoven for 48 hours to providepoly(β-carboxyl-γ-(2-mercaptoethyl)-carbamoyl-butene) 1010 as a paleyellow solid (6.15 g; 76% yield).

Polymer 1010 was then suspended in dimethyl formamide (DMF; 2 mL) andstirred for 1 hour. A mixture of2-(8-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 1020 and 476) and2-(2-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 1022 and 478) (17 mg, 23.9 micromoles) was added to thesuspension to form a reaction mixture. The reaction mixture was stirredat ambient temperature for 1 week, and was then diluted with 4 mL waterand transferred to SnakeSkin® dialysis tubing having a molecular weightcutoff (MWCO) of 7,000 daltons and containing 1 mL of water. Thedialysis tubing was closed with a clip and placed in a 2 L beakercontaining 1,600 mL water. The water was gently stirred for 2-3 daysbefore being exchanged with fresh water. Each time the water wasexchanged, a sample of the liquid in the dialysis tubing was analyzed bythin layer chromatography (TLC; silica, methanol:ethyl acetate ratio of9:1) to test for the presence of 1020 and 1022. After 3 exchanges withfresh water, the water was exchanged with a 3-5% brine solution. Thebrine was exchanged every 2-3 days, and the process repeated 6 times.The dialysis took approximately 3 weeks in total until there was nodetectable 1020 or 1022 in the liquid in the dialysis tubing. The brinewas then exchanged with water, and the stirring continued for two hours.The water was exchanged with fresh water 5 times to remove any sodiumchloride from inside the dialysis tubing. The liquid was removed fromthe dialysis tubing and used without further purification as a mixturecontaining polymer 1030.

FIG. 10B depicts chemical structures and reaction schemes for a method1050 of making a composition including a polymer having the generalformula (VII). Method 1050 includes forming polymer 1070 from a mixtureof poly(diallyl-methylamine) hydrochloride 1060,2-(8-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1020 and2-(2-(ω-bromoalkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1022. The polymer 1060 includes amine groups (R₂N—CH₃) as theplurality of nucleophilic groups bonded to the precursor polymericbackbone. Reactants 1020 and 1022 are examples of compounds 376 and 378of FIG. 3, respectively.

In one example, a mixture of2-(8-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 1020 and 476) and2-(2-(10-bromodecyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (see 1022 and 478) (20 mg, 28.1 micromoles) was weighed into a 5 mLvial and dissolved in DMSO (2 mL). Poly(diallylmethylamine)hydrochloride 1060 having a molecular weight of 20,000 daltons (100microliters of 50% solution in water, 2.5 micromoles) was added in oneportion to the mixture of 1020 and 1022 to form a reaction mixture. Thereaction mixture was stirred at ambient temperature for 1 week, and wasthen transferred to SnakeSkin® dialysis tubing having a MWCO of 7,000daltons and containing 2 mL of water. The dialysis tubing was closedwith a clip and placed in a 2 L beaker containing 1,600 mL water. Thewater was gently stirred for 2-3 days before being exchanged with freshwater. Each time the water was exchanged, a sample of the liquid in thedialysis tubing was analyzed by TLC (silica, methanol:ethyl acetateratio of 9:1) to test for the presence of 1020 and 1022. After

3 exchanges with fresh water, the water was exchanged with a 3-5% brinesolution. The brine was exchanged every 2-3 days, and the processrepeated 30 times. The dialysis took approximately 11 weeks in totaluntil there was no detectable 1020 or 1022 in the liquid in the dialysistubing. The brine was then exchanged with water, and the stirringcontinued for two hours. The water was exchanged with fresh water 5times to remove any sodium chloride from inside the dialysis tubing. Theliquid was removed from the dialysis tubing and used without furtherpurification as a mixture containing polymer 1070.

A non-leaching mediator polymer may be made by reacting one or morecompounds having general formula (IX):

and/or having general formula (X):

or salts thereof, with a compound having a plurality of polymerizablegroups. In formulas (IX) and (X), n is about 9. Examples ofpolymerizable groups include epoxide groups, isocyanate groups,carboxylic acid groups, and anhydride groups.

FIG. 11 depicts a method 1100 of making a composition including at leastone compound having the general formula (IX) or (X) and salts thereof.The method 1100 includes forming 1101 a2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-ωH-phenothiazine 1120 from a2-(ω-haloalkyl)-ωH-phenothiazine 1110, a dialkanolamine 1112, and a base1114; and forming 1102 a mixture of2-(8-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1130 and2-(2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1132 from 1120, 2-aminobenzene-1,4-disulfonic acid 1122 and Na₂S₂O₈1124. Products 1130 and 1132 are examples of compounds having thegeneral formula (IX) and (X), respectively.

Preferably the2-(8-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1130 is a2-(8-(ω-(bis(2-hydroxyethyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid, and more preferably is2-(8-(10-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid. Preferably the2-(2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1132 is a2-(2-(ω-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid, and more preferably is2-(2-(10-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid.

FIG. 11 also depicts a method 1103 of forming a polymer having apolymeric backbone and a plurality of phenothiazine groups bonded to thepolymeric backbone 1150, from a diepoxide 1140, and a mixture of2-(8-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1130 and2-(2-(ω-(bis(ω-hydroxyalkyl)amino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1132. The polymer 1150 may be, for example, a polymer having thegeneral formula (VIII). Preferably the diepoxide 1140 is adiepoxyalkane, and more preferably is 1,2,7,8-diepoxyoctane.

FIG. 12 depicts chemical structures and reaction schemes for a method1200 of a) making a composition including at least one compound havingthe general formula (IX) or (X) and salts thereof, and b) making acomposition including a polymer having the general formula (VIII).Method 1200 includes forming2-(ω-(bis-2-hydroxyethylamino)alkyl)-ωH-phenothiazine 1220 from2-(ω-bromoalkyl)-ωH-phenothiazine 1210, diethanolamine 1212, and sodiumcarbonate 1214. Reactant 1210 is an example of compound 372 of FIG. 3.Method 1200 further includes forming a mixture of2-(8-(ω-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1230 and2-(2-(ω-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1232 from the 2-(ω-(bis-2-hydroxyethylamino)alkyl)-ωH-phenothiazine1220, 2-aminobenzene-1,4-disulfonic acid 1222 and Na₂S₂O₈ 1224. Products1230 and 1232 are examples of compounds having the general formula (IX)and (X), respectively.

Method 1200 of FIG. 12 further includes forming polymer 1250 from themixture of2-(8-(ω-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1230 and2-(2-(ω-(bis-2-hydroxyethylamino)alkyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid 1232 and a diepoxide 1240.

In one example, 2-(10-bromodecyl)-ωH-phenothiazine (1210) was combinedwith diethanolamine 1212 (1.1 equivalents), and sodium carbonate 1214(2.2 equivalents) in acetonitrile to form a first reaction mixture, andthe first reaction mixture was heated at reflux overnight. An aqueousworkup of the reaction mixture yielded2-(10-(bis-2-hydroxyethylamino)decyl)-ωH-phenothiazine (1220) in nearlyquantitative yield (97% purity by high-performance liquid chromatography(HPLC)). The product was added to a solution of2-aminobenzene-1,4-disulfonic acid 1222 and Na₂S₂O₈ 1224 in a mixture ofTHF and water to form a second reaction mixture, and product was presentby liquid chromatograph/mass spectrometry (LCMS) after 30 minutes atroom temperature. The second reaction mixture was poured into asaturated aqueous sodium bicarbonate solution, yielding a precipitatethat was isolated by filtration. The precipitate was determined to be amixture of2-(8-(10-(bis-2-hydroxyethylamino)decyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (1230) and2-(2-(10-(bis-2-hydroxyethylamino)decyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (1232) by LCMS. The purity of the mixed product was 83%, with atotal yield of 33%.

The mixture of2-(8-(10-(bis-2-hydroxyethylamino)decyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (1230) and2-(2-(10-(bis-2-hydroxyethylamino)decyl)-3H-phenothiazin-3-ylideneamino)benzene-1,4-disulfonicacid (1232) was dissolved in THF and treated with sodium hydride (NaH,2.2 equivalents) and stirred for 10 minutes at room temperature. Thediepoxide (1240) 1,2,7,8-diepoxyoctane (1.1 equivalents) was then addedto form a third reaction mixture, which was heated at 55° C. for oneweek. Analysis of a sample of the third reaction mixture by TLC showed abaseline spot having a low intensity, the continued presence of thestarting material, and some streaks close to the starting material. Thethird reaction mixture was heated to 55° C. again for an additionalweek, and subsequent analysis by TLC was similar to the first analysis,except that the baseline spot was slightly more intense. The thirdreaction mixture was then heated to reflux for one week, and subsequentanalysis by TLC showed no change from the previous analysis. Theprecipitate was isolated and dissolved in water, yielding a red aqueoussolution and a residual insoluble precipitate. The aqueous solution wasbelieved to contain polymer 1250.

A non-leaching mediator polymer may be made by reacting a polymer havinga precursor polymeric backbone and a plurality of reactive functionalgroups bonded to the precursor polymeric backbone, with a compoundhaving a first functional group that may be oxidized or reduced andhaving a second functional group that can react with the reactivefunctional groups bonded to the precursor polymeric backbone. Thecompound having a first functional group that may be oxidized or reducedand having a second functional group that can react with the reactivefunctional groups bonded to the precursor polymeric backbone may be apolymer bondable mediator. A polymer bondable mediator includes amediator that can be bonded to a polymer.

Compounds having the general formulas (I), (II) and/or (III) may beuseful as polymer bondable mediators. The terminal halogen group may bereacted with a functional group of a polymer, bonding the mediator tothe polymer. Bonding includes covalent bonding where an electron pair isshared between two atoms. The terminal halogen group may be convertedinto a different functional group, and this modified mediator may thenbe bonded to a polymer. The polymer may be a binder of a reagentcomposition for an electrochemical biosensor.

FIG. 13 depicts examples of polymer bondable mediators having terminalfunctional groups other than halogen. These examples are based oncompounds having the general formula (I); however, similar derivativesof compounds having the general formulas (II) or (III) are envisioned.The exemplary terminal functional groups depicted in FIG. 13 are,clockwise from top, hydroxyl, aldehyde, ester (R¹=organic group),carboxylic acid, amine, amide, azide and alkyne groups.

A non-leaching mediator polymer may be made by polymerizing monomers,where the monomers include a compound having a first functional groupthat may be oxidized or reduced and having a second functional groupthat can participate in a polymerization or copolymerization reaction.The compound having a first functional group that may be oxidized orreduced and having a second functional group that can participate in apolymerization or copolymerization reaction may be a polymerizablemediator. A polymerizable mediator includes a mediator that canpolymerize to form a polymer, or that can copolymerize with othermonomers to form a copolymer.

Compounds having the general formulas (I), (II) and/or (III) may beuseful as intermediates for preparing polymerizable mediators. Theterminal halogen group may be converted into a functional group capableof polymerization or copolymerization. For example, the terminal halogenmay be converted into a carbon-carbon double or triple bond. Thismodified mediator having a terminal unsaturated group may thenpolymerize or copolymerize through radical, anionic and/or cationicpolymerization. The terminal halogen also may be converted into afunctional group that can undergo a condensation reaction. This modifiedmediator having a terminal functional group may then polymerize orcopolymerize through a condensation polymerization. The resultingpolymer may be used as a binder of a reagent composition for anelectrochemical biosensor.

FIG. 14 depicts examples of polymerizable mediators having terminalfunctional groups other than halogen. These examples are based oncompounds having the general formula (I); however, similar derivativesof compounds having the general formulas (II) or (III) are envisioned.The exemplary terminal functional groups depicted in FIG. 14 are,clockwise from top, alkene, alkyne, acrylate, methacrylate, epoxide,phenol, isocyanate and silanol groups (R², R³=organic groups).

The examples depicted in FIG. 14 also may be used as polymer bondablemediators, depending on the functional groups bonded to the precursorpolymeric backbone. Likewise, the examples depicted in FIG. 13 also maybe used as polymerizable mediators, depending on the polymerizationreaction conditions and/or on the copolymerization reactivity of othermonomers. In one example, a mediator having a terminal alkyne group isdepicted in both FIG. 13 and in FIG. 14. In another example, a compoundhaving the general formula (IX) or (X) is a species of the mediatorhaving a terminal amine group depicted in FIG. 13.

Compounds having the general formulas (I), (II) and/or (III) also may beuseful as surface active mediators. A surface active mediator is anon-bonded mediator that includes a hydrophobic portion and ahydrophilic portion. For compounds having the general formulas (I), (II)and/or (III), the alkyl chain group may function as the hydrophobicportion, whereas the benzene 1,4-disulfonic acid group may function asthe hydrophilic portion. The compounds also may be modified byconverting the terminal halogen group into a more hydrophobic group,such as an alkyl group, to further increase the difference in solubilityof the two portions of the mediators.

Polymer bondable mediators, polymerizable mediators and/or surfaceactive mediators may be used to provide immobilized mediators forelectrochemical bioanalysis. While the benzene 1,4-disulfonic acid groupof the compound may be sufficiently solubilized in an aqueous sample tointeract with an analyte and/or an enzyme, the alkyl chain group of thecompound may have a solubility in the sample that is so low as toinhibit the entire compound from dissolving in the sample. The overallsolubility of the mediator may be further diminished when the mediatoris bonded to a polymer. Thus, compounds having the general formulas (I),(II) and/or (III), derivatives of these compounds bonded to a polymer,polymers having a polymeric backbone and a plurality of phenothiazinegroups bonded to the polymeric backbone, and/or polymers havingstructure (VI) or (VII) may be useful as mediators having little or noability to leach into a sample.

While various embodiments of the invention have been described, it willbe apparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.Accordingly, the invention is not to be restricted except in light ofthe attached claims and their equivalents.

What is claimed is:
 1. An electrochemical test sensor comprising aworking electrode, a counter electrode and a mediator, the mediatorincluding a polymer, the polymer including a polymeric backbone, and aplurality of phenothiazine groups bonded to the polymeric backbone;where the plurality of phenothiazine groups includes at least one of aphenothiazine group having the general formula (IV):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, and a phenothiazinegroup having the general formula (V):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded.
 2. Theelectrochemical test sensor of claim 1, wherein the phenothiazine groupis bonded to the polymeric backbone through a carbon-heteroatom bond. 3.The electrochemical test sensor of claim 1, wherein the phenothiazinegroup is bonded to the polymeric backbone through a group selected fromthe group consisting of an ether group, an ester group, a carbonategroup, a urea group, a urethane group, an amine group, an amide group, athioether group, a thiourea group and a sulfonamide group.
 4. Theelectrochemical test sensor of claim 3, wherein the phenothiazine groupis bonded to the polymeric backbone through a group selected from anether group or an ester group.
 5. The electrochemical test sensor ofclaim 3, wherein the phenothiazine group is bonded to the polymericbackbone through a carbonate group.
 6. The electrochemical test sensorof claim 3, wherein the phenothiazine group is bonded to the polymericbackbone through a urea group or a urethane group.
 7. Theelectrochemical test sensor of claim 3, wherein the phenothiazine groupis bonded to the polymeric backbone through an amine group or an amidegroup.
 8. The electrochemical test sensor of claim 3, wherein thephenothiazine group is bonded to the polymeric backbone through athioether group or a thiourea group.
 9. The electrochemical test sensorof claim 3, wherein the phenothiazine group is bonded to the polymericbackbone through a sulfonamide group.
 10. The electrochemical testsensor of claim 1 further including glucose oxidase.
 11. Theelectrochemical test sensor of claim 1 further including glucosedehydrogenase.
 12. An electrochemical test sensor comprising a workingelectrode, a counter electrode, glucose dehydrogenase, and a mediator,the mediator including a polymer, the polymer including a polymericbackbone, and a plurality of phenothiazine groups bonded to thepolymeric backbone; where the plurality of phenothiazine groups includesat least one of a phenothiazine group having the general formula (IV):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, and a phenothiazinegroup having the general formula (V):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, wherein thephenothiazine group is bonded to the polymeric backbone through acarbon-heteroatom bond.
 13. An electrochemical test sensor comprising aworking electrode, a counter electrode, glucose oxidase, and a mediator,the mediator including a polymer, the polymer including a polymericbackbone, and a plurality of phenothiazine groups bonded to thepolymeric backbone; where the plurality of phenothiazine groups includesat least one of a phenothiazine group having the general formula (IV):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, and a phenothiazinegroup having the general formula (V):

and salts thereof, where n is about 9 and “R” represents the polymericbackbone to which the phenothiazine group is bonded, wherein thephenothiazine group is bonded to the polymeric backbone through acarbon-heteroatom bond.